Desensitization to pharmacological agents has important clinical relevance in the treatment of various diseases. The molecular events that underlie desensitization have not been completely elucidated but are likely to involve modification of signaling proteins, particularly receptors. In the superfamily of G- protein coupled receptors (GPRs), desensitization appears to involve regulation of the receptors by G-protein uncoupling. This proposal will focus on the molecular basis of desensitization of GPRs of two related melatonin receptors, mt1 and mt2. The mt1 melatonin receptors attenuate adenylyl cyclase and are primarily expressed in the brain. The mt2 melatonin receptors also attenuate adenylyl cyclase and are primarily expressed in the retina. Preliminary data demonstrate that agonist exposure results in the desensitization of both the mt1 and mt2 melatonin receptors. In addition, melatonin exposure dramatically alters tubulin assembly in CHO cells transfected with the mt1 melatonin receptor. The hypothesis that underlies this proposal is that melatonin receptor/G-protein uncoupling due to impaired GDP/GTP exchange mechanisms and/or to enhanced GTPase activity is a critical event in desensitization. The long term objectives of my laboratory are to understand the mechanisms underlying G-protein coupled receptor function and regulation. This proposal will determine the role that G-proteins play in GPR function and regulation through the following three specific objectives: 1) We will use immunoblot analysis, G-shift, GTPase hydrolysis and GTP exchange assays to compare and contrast the function of G-proteins in CHO cells transfected with either the mt1 (mt1-CHO) or mt2 (mt2-CHO) melatonin receptor, 2) We will use immunoblot analysis, G-shift, GTP hydrolysis and GTP exchange assays to compare and contrast the effects of agonist exposure on G-protein function in mt1-CHO and mt2-CHO cells, and 3) We will determine the effects of putative regulators of G-protein function, that is, tubulin and RGS4, on G-protein function using anti-sense knock down, immunoblot, GTP exchange and GTP hydrolysis assays. The results of the proposed experiments should provide a model of GPR desensitization by G-proteins and will provide a novel approach to treating various disease states associated with a loss of GPR function following agonist exposure.